One of the pervasive challenges in landscape genetics is detecting gene flow patterns within continuous populations of highly mobile wildlife. Understanding population genetic structure within a continuous population can give insights into social structure, movement across the landscape and contact between populations, which influence ecological interactions, reproductive dynamics, or pathogen transmission. We investigated the genetic structure of a large population of deer spanning the area of Wisconsin and Illinois, USA, affected by chronic wasting disease. We combined multi-scale investigation, landscape genetic techniques and spatial statistical modeling to address the complex questions of landscape factors influencing population structure. We sampled over 2,000 deer and used spatial autocorrelation and a spatial principal components analysis to describe the population genetic structure. We evaluated landscape effects on this pattern using a spatial auto-regressive model within a model selection framework to test alternative hypotheses about gene flow. We found high levels of genetic connectivity, with gradients of variation across the large continuous population of white-tailed deer. At the fine scale, spatial clustering of related animals was correlated with the amount and arrangement of forested habitat. At the broader scale, impediments to dispersal were important to shaping genetic connectivity within the population. We found significant barrier effects of individual state and interstate highways and rivers. Our results offer an important understanding of deer biology and movement that will help inform the management of this species in an area where over-abundance and disease spread are primary concerns.